1. Field of the Invention
The present invention generally relates to the fields of cytokines and immunology. More specifically, it concerns the surprising delineation of the immunosuppressive and immunostimulatory properties of the molecule, IL-10. The invention thus provides mammalian and human IL-10 biological compositions that have only immunosuppressive properties, and are not comprised by immunostimulatory effects. The new IL-10 constructs may thus be used in various in vitro and in vivo methods, particularly in immunosuppressive therapies, and combinations thereof, for various inflammatory diseases and disorders, and in transplantation.
2. Description of Related Art
IL-10 was originally described as cytokine synthesis inhibitory factor (Fiorentino et al., 1989) because of its ability to turn off cytokine production by T cells. IL-10 is now known to have both immunostimulatory and immunosuppressive effects, which may vary depending on the cell types involved and other events in immune regulation.
Early in vitro studies showed that IL-10 can directly inhibit TH1 and TH2 IL-2 production (de Waal et al., 1993) and IL-5 production (Schandene et al., 1994) at the level of the T cell. T cell stimulation in vitro in the presence of IL-10 can lead both to long term anergy (Groux et al., 1996) and the production of a negative regulatory T cell subset (Groux et al., 1997). In vivo, IL-10 inhibits T cell mediated delayed type hypersensitivity and contact hypersensitivity (Powrie et al., 1993; Enk et al., 1994; Ferguson et al., 1994; Li et al., 1994; Berg et al., 1995; Flores-Villanueva et al., 1996).
Further investigations demonstrated that the immunosuppressive effects of. IL-10 are more often at the level of the APC and not directly at the level of the T cell (Fiorentino et al., 1991a). Thus, IL-10 inhibits monocyte and macrophage synthesis of IL-lxcex1, IL-xcex2, IL-6, IL-8, IL-12, TNFxcex1, GM-CSF, and reactive oxygen and nitrogen intermediates (de Waal et al., 1991a; Bogdan et al., 1991; Fiorentino et al., 1991b; D""Andrea et al., 1993). IL-10 inhibits dendritic cell stimulation of TH1 IFNxcex3 production (Macatonia et al., 1993); APC B7 expression (Ding et al., 1993; Willems et al., 1994; Villanueva et al., 1994); and antigen presentation to TH1 but not TH2 cells (Enk et al., 1993), while inducing IL-1 receptor antagonist production in neutrophils (Cassatella et al., 1994).
IL-10 also suppresses epidermal Langerhans cell APC functions (Chang et al., 1994; Beissert et al., 1995), chemokine expression by monocytes (Berkman et al, 1995), and the bactericidal response of macrophages to IFNxcex3 (Murray et al., 1997). IL-10 treated dendritic cells induce peptide antigen and alloantigen specific tolerance (Steinbrink et al., 1997). Additional studies demonstrated that IL-10 inhibits the immune function of other cell types, too. Thus, IL-10 inhibits NK cell production of IFNxcex3 (Tripp et al., 1993), ICAM-1 expression on activated vascular endothelial cells (Eissner et al., 1996), and T independent responses of B cells (Pecanha et al., 1993).
Therefore, investigations have shown that the predominant effect of IL-10, is to suppress multiple immune responses through individual actions on T cells, B cells, APCs, and other cell types. The prominent effects on IL-12 and IFNxcex3 production and responsiveness suggest that IL-10 channels immunity away from TH1 and toward TH2 responses, although both types of responses can be inhibited under some circumstances (de Waal et al., 1993; Schandene et al., 1994; Fiorentino et al., 1991a; Macatonia et al., 1993; Enk et al., 1993; Steinbrink et a., 1997). It is not clear why IL-10 has opposing effects on TH2 subsets, inhibiting or promoting subset activity.
Additional correlative studies have focused on the role of IL-10 in various disease states or models. IL-10 can effectively treat the cytokine syndrome and toxicity caused by anti-CD3
MAb or endotoxin by inhibiting the production of proinflammatory cytokines (Wissing et al., 1997; Howard et al., 1993; Pajkrt et al., 1997). Autoimmune models of rheumatoid arthritis (Katsikis et al., 1994), thyroiditis (Mignon-Godefroy et al., 1995), and collagen-induced arthritis (Kasama et al., 1995) and a model of herpetic stromal keratitis (Daheshia et al., 1997); all suggest negative regulatory roles for IL-10 in limiting inflammation and immunopathology.
IL-10 can inhibit tumor immunity (Qin et al., 1997); and there are numerous examples of a relation among IL-10 expression, allograft survival, and decreased alloreactivity (Gorczynski and Wojcik, 1994; Bacchetta et al., 1994; Mottram et al., 1995; Mutsuda et al., 1994; Pxc3xa9guet-Navarro et al., 1994; Danzer et al., 1994; Burke et al., 1995; Sayegh et al., 1995; Gorczynski et al., 1995). IL-10 deficient knockout mice have highly polarized TH1 responses and develop a severe colitis related to chronic stimulation by enteric antigens (Rennick et al., 1997). In humans, Crohn""s colitis may even be susceptible to treatment with systemically administered IL-10 (van Deventer et al., 1997). Likewise, psoriasis may be due to unregulated cutaneous TH1 responses and IL-10 administration may be effective treatment for humans (Asadulla et al., 1998).
Further reinforcement for the notion that IL-10 is an immunosuppressive cytokine came from the discovery of vIL-10 (Moore et al., 1990), which was shown to have identical immunosuppressive properties to cIL-10, inhibiting IFNxcex3 production (Hsu-et al., 1990), MHC class II expression (de Waal et al., 1991b), T cell proliferation (Del Prete et al., 1993), and B cell IgE production (Punnonen et al., 1993). There is speculation that vIL-10 is important for EBV pathogenesis through suppression of the specific anti-viral immune response. A recent case report provides circumstantial evidence for this view (Nast et al., 1997).
Despite the acceptance of IL-10 as an immunosuppressive molecule, a number of findings suggest that IL-10 has actions which are more complex than originally proposed. IL-10 can inhibit T independent B cell responses, but not T dependent responses (Pecanha et al., 1993).
In fact, IL-10 can act as a B cell growth factor (Fei et al., 1990), a property shared by cIL-10 and vIL-10, and even support the autocrine growth of B cell lymphomas (Beatty et al., 1997). IL-10 can act as a proliferative co-factor for immature and mature thymocytes stimulated by IL-2 plus IL-4,(MacNeil et al., 1990). Interestingly, while cIL-10 can co-stimulate thymocyte proliferation and B cell MHC class II expression, vIL-10 cannot (Fei et al., 1990; MacNeil et al., 1990), suggesting differences in the structure and function of the two molecules.
IL-10 genes transfected into ovarian or mammary tumors promote anti-tumor immunity and rejection, instead of suppressing the immune response (Richter et al., 1993; Allione et al., 1994). Most significantly, transduction of tumors with a retroviral vector encoding mIL-10 results in enhanced tumor immunity and rejection, while vIL-10 tumor cell transduction results in immune suppression and tumor growth (Suzuki et al., 1995). Studies using a cardiac allograft model also showed that vIL-10 prolongs graft survival while mIL-10 impairs graft survival (Qin et al., 1996a). These results demonstrate that cIL-10 is not necessarily exclusively immunosuppressive in its actions, and that vIL-10 is immunosuppressive under conditions in which cIL-10 is immunostimulatory.
There are a large number of studies in which there has been a general failure to correlate the presence or absence of IL-10 with allograft survival or rejection (Baan et al., 1994; Shirwan et al., 1994; Cunningham et al., 1994; Le Moine et al., 1994; Sun et al., 1994; Allen et al., 1993; Garlisi et al., 1993; Bishop et al., 1993; Merville et al., 1993; Delvaux et al., 1994; Merville et al., 1995; Krenger et al., 1994). One interpretation of these results is that the presence or absence of other cytokines such as IL-4, IFNxcex3, or IL-12 could affect the final immune outcome. Another view, however, is that IL-10 could be acting in a proinflammatory fashion and actually contributing to graft rejection. Indeed IL-10 can induce the expression of E-selectin on vascular endothelium (Vora et al., 1996), which would be expected to promote and sustain inflammatory responses.
Likewise, the TH2 polarization induced by IL-10 enhances the development of granulomata and chronic inflammation (Wynn et al., 1997). IL-10 stimulates the development of systemic autoimmune disease in NZB/W F1 mice, which is mediated primarily by B cells, while anti-IL-10 mAb delays the onset of autoimmunity (Ishida et al., 1994). IL-10 inhibits CD4+, but promotes CD8+ T lymphocyte migration (Jinquan et al., 1993), and enhances the development of tumor specific B cells and CD8- cytotoxic T lymphocyte (CTL) responses in vivo (Giovarelli et al., 1995). These studies, conducted primarily in in vivo models, all suggest proinflammatory functions for IL-10 under some circumstances. However, those studies do not define the cellular or molecular variables that determine immunosuppressive versus immunostimulatory responses.
Work in autoimmune and alloimmune diabetes models has generated some of the most dichotomous results with respect to IL-10 immunologic activities. Administration of IL-10 can prevent the development of autoimmune diabetes (Zheng et al., 1997) and prolong syngeneic islet survival in autoimmune diabetic recipients (Rabinovitch et al., 1995). Correlative studies show a decrease in endogenous IL-10 expression in T cells of spontaneously diabetic animals (Sarukhan et al. 1998), but increased IL-10 in anergized T cells (Buer et al.,. 1998). In fact, TH1 clones that adoptively transfer autoimmune diabetes to normal recipients can be, rendered ineffective by retroviral transduction with a vector encoding mIL-10 (Moritani et al., 1996).
Similar adoptive transfer results have also been obtained with IL-10 transduced T cell clones in experimental autoimmune encephalomyelitis (Mathisen et al., 1997) and Leishmania infection (Hagenbaugh et al., 1997) models. However, in another model, IL-10 administration not only failed to prolong islet allograft survival, but also accelerated islet destruction and increased granzyme B gene expression, suggesting a role for IL-10 in CTL induction (Zheng et al., 1995). Sarvetnick and colleagues generated mice with a. mIL-10 transgene regulated by an insulin promoter (Wogensen et al., 1993). The pancreata of these mice had a pronounced leukocytic infiltrate of CD4+ and CD8+ T cells, B cells, and macrophages, along with activation of the vascular endothelium. Transgenic IL-10 expression in these mice did not prevent or delay autoimmune or alloimmune disease (Lee et al., 1994; Wogensen et al., 1994).
In sum, the foregoing studies all show that IL-10 may have immunostimulatory or immunosuppressive effects depending on the assay, cell types involved, or other concomitant immune events. Unfortunately, the molecular and cellular basis for this dichotomy is not currently defined. The ability to manipulate responses to IL-10 in either a stimulatory or suppressive direction would be import, in determining what aspects of IL-10 activity are important for normal T cell development and channeling TH1 and TH2 responses, and would be of enormous practical value in regulating immune responses, e.g., for use in for disease therapy.
The present invention overcomes these and other drawbacks inherent in the prior art with the surprising discovery of the molecular basis for the immunosuppressive and immunostimulatory properties of IL-10. The invention particularly embodies the discovery that a single amino acid difference in IL-10 determines the immunological activity of the entire molecule.
Despite earlier speculations that the key differences were likely to lie in the N- or C-terminal regions of the IL-10 molecule, based upon a comparison of the cIL-10 and vIL-10 molecular structures, the present inventors show that the key amino acid lies in the central third of the molecule. In particular, the inventors discovered that the relatively conservative change of isoleucine for alanine, at position of 87 of the native molecule, was sufficient to produce an IL-10 molecule with only immunosuppressive properties, rather than the mixed immunomodulatory effects of the native mammalian IL-10s.
The present invention thus provides mammalian, including human, immunosuppressive IL-10 compositions either alone or in combination with other agents, and various in vitro and in vivo methods of using such compositions and combinations thereof. Uses include immunosuppressive and combination therapies for a number of diseases and disorders related to inflammation, including inflammatory bowel disease, grafts and transplantation, fibrosis, scarring and tumor treatment.
The invention first provides a composition comprising at least one mutant IL-10 polypeptide, comprising a substantially mammalian IL-10 amino acid: sequence wherein isoleucine at position 87 of the mature polypeptide is replaced by at least one amino acid other than leucine or valine. In certain aspects, the at least one amino acid is alanine or glycine. In other aspects, the composition comprises at least a first substantially purified mutant IL-10 polypeptide. In further aspects, the polypeptide lacks a signal sequence, as represented in FIG. 1.
In some embodiments, the composition comprises a substantially bovine, murine or porcine amino acid sequence. In particular embodiments, the composition comprises a substantially murine IL-10 amino acid sequence. In additional aspects, the composition comprises the sequence of SEQ ID NO:2. In other aspects, the isoleucine at position 87. of the mature polypeptide is replaced by alanine.
In other embodiments, the composition comprises a substantially human IL-10 amino acid sequence. In particular aspects, the composition comprises the sequence of SEQ ID NO: 1. In further aspects, the isoleucine at position 87 of the mature polypeptide is replaced by alanine. In other aspects, the composition comprises the amino acid sequence of SEQ ID NO:4.
In certain embodiments, the composition comprises a chimera of substantially mammalian IL-10 amino acid sequences.
In additional embodiments, the composition is prepared by recombinant expression. In further embodiments, the composition is operatively attached to a selected amino acid sequence to form a fusion protein.
In some embodiments, the composition is dispersed in a pharmaceutically acceptable formulation. In certain aspects, the polypeptide is a therapeutic agent. In additional aspects, the pharmaceutically acceptable formulation further comprises at least a second therapeutic agent. In specific aspects, the polypeptide is an immunosuppressive agent. In further aspects, the composition comprises a biologically effective amount of at least a second immunosuppressive agent. In particular aspects, the composition further comprises a biologically effective amount of corticosteroid, sulfasalazine, cyclosporin A, mercaptopurine, azathioprine or a combination thereof. In additional aspects, the composition further comprises a biologically effective amount of tacrolimus, sirolimus, mycophenolate mofetil or a combination thereof. In some aspects, the composition further comprises a biologically effective amount of an immunosuppressive antiserum, immunosuppressive antibody or a combination thereof. In other aspects, the composition further comprises a biologically effective amount of an immunosuppressive antisera ATG, Atgam, Thymoglobulin, immunosuppressive antibody OKT3 or a combination thereof. In additional aspects, the composition further comprises a biologically effective amount of IL-4.
In specific embodiments, the composition may be formulated for parenteral administration. In some aspects, the composition is formulated for intravenous injection.
The invention also provides a composition comprising at least one mutant IL-10 polypeptide that comprises a substantially mammalian IL-10 amino acid sequence wherein isoleucine at position 87 of the mature polypeptide is replaced by alanine or glycine. In certain embodiments the composition comprises at least a one substantially purified mutant IL-10 polypeptide.
The invention provides a composition comprising at least one mutant IL-10 polypeptide, comprising a substantially mammalian IL-10 amino acid sequence wherein isoleucine at position 87 of the mature polypeptide is replaced by at least one amino acid other than leucine or valine.
The invention provides at least one mutant IL-10 polypeptide, comprising a substantially mammalian IL-10 amino acid sequence wherein isoleucine at position 87 of the polypeptide that lacks a signal sequence, as represented in FIG. 1, is replaced by alanine or glycine.
The invention provides at least one mutant IL-10 polypeptide, comprising a substantially mammalian IL-10 amino acid sequence wherein isoleucine at position 87 of the mature polypeptide is replaced by alanine or glycine. In certain embodiments, the mutant IL-10 polypeptide(s) comprises a substantially mammalian IL-10 amino acid sequence wherein isoleucine at position 87 of the mature polypeptide is replaced by alanine. In other embodiments, the mutant IL-10 polypeptide(s) comprises a substantially bovine or porcine IL-10 amino acid sequence wherein isoleucine at position 87 of the mature polypeptide(s) is replaced by alanine or glycine.
In some embodiments, the mutant IL-10 polypeptide(s) comprises a substantially murine IL-10 amino acid sequence wherein isoleucine at position 87 of the mature polypeptide(s) is replaced by alanine or glycine. In certain aspects, the mutant IL-10 polypeptide(s) comprises the substantially murine IL-10 amino acid sequence of SEQ ID NO:2, wherein isoleucine at position 87 of the mature polypeptide(s) is replaced by alanine or glycine. In further aspects, the mutant IL-10 polypeptide(s) comprises the substantially murine IL-10 amino acid sequence of SEQ ID NO:2, wherein isoleucine at position 87 of the mature polypeptide(s) is replaced by alanine.
In certain embodiments, the mutant IL-10 polypeptide(s) comprises a substantially human IL-10 amino acid sequence wherein isoleucine at position 87 of the mature polypeptide(s) is replaced by alanine or glycine. In certain aspects, the mutant IL-10 polypeptide(s) comprises the substantially human IL-10 amino acid sequence of SEQ ID NO: 1, wherein isoleucine at position 87 of the mature polypeptide(s) is replaced by alanine or glycine. In further aspects, the mutant IL-10 polypeptide(s) comprises the substantially human IL-10 amino acid sequence of SEQ ID NO:1, wherein isoleucine at position 87 of the mature polypeptide(s) is replaced by alanine. In additional aspects, the mutant IL-10 polypeptide(s) comprises the amino acid sequence of SEQ ID NO:4.
In certain embodiments, the mutant IL-10 polypeptide(s) comprises a chimera of substantially mammalian IL-10 amino acid sequences wherein isoleucine at position 87 of the mature polypeptide(s) is replaced by alanine or glycine. In certain aspects, the mutant IL-10 polypeptide(s) comprises a chimera of substantially murine and human IL-10 amino acid sequences, wherein isoleucine at position 87 of the mature polypeptide(s) is replaced by alanine or glycine.
In certain embodiments, the mutant IL-10 polypeptide(s) may be prepared by recombinant expression. In other embodiments, the mutant IL-10 polypeptide(s) may be operatively attached to at least one selected amino acid sequence to form at least one fusion protein.
In additional embodiments, the mutant IL-10 polypeptide(s) may be dispersed in one or more pharmaceutically acceptable formulation(s). In certain aspects, the pharmaceutically acceptable formulation(s) further comprises at least a second therapeutic agent. In additional aspects, the formulations(s) further comprise at least a third, at least a fourth, at; least a fifth, comprise at least a sixth, at least a seventh or more therapeutic agents.
The invention provides at least one non-viral IL-10 polypeptide variant, comprising a substantially non-viral IL-10 amino acid sequence that comprises either alanine or glycine at position 87 of the mature polypeptide(s).
The invention provides at least one immunosuppressive IL-10 polypeptide, comprising a substantially mammalian IL-10 amino acid sequence wherein isoleucine at position 87 of the mature polypeptide(s) is replaced by alanine or glycine.
The invention provides at least one IL-10 derivative, comprising a substantially mammalian IL-10 amino acid sequence wherein isoleucine at position 87 of the mature polypeptide is replaced by an amino acid other than leucine or valine, the IL-10 derivative exerting at least one immunosuppressive effect on one or more T cell(s), B cell(s) or antigen presenting cell(s). In particular embodiments, the IL-10 derivative comprises a substantially mammalian IL-10 amino acid sequence wherein isoleucine at position 87 of the mature polypeptide is replaced by alanine or glycine.
The invention provides at least one IL-10 polypeptide essentially devoid of immunostimulatory activity, comprising a substantially mammalian IL-10 amino acid sequence wherein isoleucine at position 87 of the mature polypeptide(s) is replaced by alanine or glycine.
The invention provides at least one IL-10 derivative, comprising a substantially mammalian IL-10 amino acid sequence wherein isoleucine at position 81 of the mature polypeptide(s) is replaced by alanine or glycine, the IL-10 derivative essentially lacking one or more immunostimulatory effect(s) on one or more T cell(s), B cell(s) or antigen presenting cell(s).
The invention provides at least one IL-10 derivative that retains at least one pluripotent immunosuppressive activity in the absence of immunostimulatory activity, the IL-10 derivative comprising a substantially mammalian IL-10 amino acid sequence wherein isoleucine at position 87 of the mature polypeptide(s) is replaced by alanine or glycine.
The invention provides at least one nucleic acid segment comprising at least a first isolated coding region that encodes at least one mutant IL-10 polypeptide that comprises a substantially mammalian IL-10 amino acid sequence wherein isoleucine at position 87 of the mature polypeptide(s) is at least one amino acid other than leucine or valine. In certain specific embodiments, the at least one amino acid is alanine or glycine.
In certain embodiments, the isolated coding region encodes at least one human mutant IL-10 polypeptide comprising the amino acid sequence of SEQ ID NO:1, wherein isoleucine at position 87 is replaced by alanine or glycine. In certain aspects, the isolated coding region encodes at least one human mutant IL-10 polypeptide comprising the amino acid sequence of SEQ ID NO:4. In further aspects, the nucleic acid segment(s) is at least one RNA segment or DNA segment. In further aspects, the nucleic acid segment(s) is positioned under the control of at least one promoter, including but not limited to at least one CMV promoter. In other aspects, the nucleic acid segment(s) may be further defined as one or more recombinant vector(s). In certain aspects, the nucleic acid segment(s) may be comprised within a recombinant host cell.
The invention provides at least one recombinant vector comprising at least a first expression unit that expresses at least one mutant IL-10 polypeptide that comprises a substantially mammalian IL-10 amino acid sequence wherein isoleucine at position 87 of the mature polypeptide is replaced by alanine or glycine. In certain embodiments, the recombinant vector(s) may be further defined as at least one recombinant viral vector. In certain aspects, the recombinant vector(s) may be further defined as at least one recombinant viral vector comprised within at least one recombinant virus particle or virion.
The invention provides at least one host cell comprising at least a first exogenous DNA segment that encodes at least one mutant IL-10 polypeptide that comprises a substantially mammalian IL-10 amino acid sequence wherein isoleucine at position 87 of the mature polypeptide is replaced by alanine or glycine. In certain embodiments, the host cell(s) is at least one prokaryotic host cell or at least one eukaryotic host cell. In certain aspects, the DNA is segment(s) is introduced into the cell(s) by means of at least one recombinant vector. In further aspects, the host cell(s) expresses the DNA segment(s) to produce the encoded mutant IL-10 polypeptide(s). In additional aspects, the host cell(s) is comprised within at least one animal.
The invention also provides a method of using at least one mutant IL-10 DNA segment, comprising expressing the DNA segment(s) in at least one recombinant host cell and collecting the mutant IL-10 expressed by the cell(s); wherein the mutant IL-10 DNA segment comprises at least a first isolated coding region that encodes at least one mutant IL-10 polypeptide that comprises a substantially mammalian IL-10 amino acid sequence wherein isoleucine at position 87 of the mature polypeptide is replaced by alanine or glycine. In certain aspects, the mutant IL-10 expressed by the cell(s) is collected by at least one process comprising at least one column chromatography step.
The invention provides at least one pharmaceutical composition comprising a biologically effective amount of at least a first mutant IL-10 polypeptide that comprises a substantially mammalian IL-10 amino acid sequence wherein isoleucine at position 87 of the mature polypeptide is replaced by alanine or glycine, or a biologically effective amount of at least one nucleic acid segment or vector that encodes the mutant IL-10 polypeptide(s). In certain embodiments, the pharmaceutical composition(s) comprises a biologically effective amount of at least a first mutant IL-10 polypeptide, or at least one nucleic acid segment or vector that encodes the mutant IL-10 polypeptide(s).
In other embodiments, the pharmaceutical composition(s) further comprises a biologically effective amount of at least a second therapeutic agent. In certain aspects, the pharmaceutical composition(s) further comprises a biologically effective amount of at least a second immunosuppressive agent. In additional aspects, the compositions(s) further comprise at least a third, at least a fourth, at least a fifth, comprise at least a sixth, at least a seventh or more agents. In additional aspects, the pharmaceutical composition further comprises a biologically effective amount of one or more corticosteroid, sulfasalazine, cyclosporin A, mercaptopurine, azathioprine, tacrolimus, sirolimus, mycophenolate mofetil, an immunosuppressive antiserum, immunosuppressive antibody or a combination thereof the aformentioned agents. In further aspects, the pharmaceutical composition(s) further comprises a biologically effective amount of one or more of the following agents, the immunosuppressive antisera ATG, Atgam, Thymoglobulin or the immunosuppressive antibody OKT3. In additional embodiments, the pharmaceutical composition(s) further comprises a biologically effective amount of IL-4.
In certain embodiments, the pharmaceutical composition(s) may be formulated for parenteral administration. In certain aspects, the pharmaceutical composition(s) may be formulated for intravenous injection.
The invention provides one or more therapeutic kit(s) comprising, in at least a first suitable container, a combined effective amount of at least one mutant IL-10 polypeptide that comprises a substantially mammalian IL-10 amino acid sequence wherein isoleucine at position 87 of the mature polypeptide is replaced by alanine or glycine; and at least a second therapeutic agent. In additional aspects, the kit(s) further comprise at least a third, at least a fourth, at least a fifth, comprise at least a sixth, at least a seventh or more therapeutic agents. In certain embodiments, the mutant IL-10 polypeptide(s) and the therapeutic agent(s) are comprised within a single container. In other embodiments, the mutant IL-10 polypeptide(s) and the at least a therapeutic agent(s) are comprised within distinct container(s). In additional embodiments, the at least one of the mutant IL-10 polypeptide and the therapeutic agent(s) are dispersed within one or more pharmaceutically acceptable formulation(s). In further embodiments, the at least one of the mutant IL-10 polypeptide and the therapeutic agent(s) are in at least one lyophilized form. In certain aspects, the kit(s) further comprises at least a second container comprising a pharmaceutically acceptable diluent.
In particular embodiments, the therapeutic agent(s) is at least one immunosuppressive agent. In certain aspects, the therapeutic agent(s) is one or more of the following: a corticosteroid, sulfasalazine, cyclosporin A, mercaptopurine, azathioprine, tacrolimus, sirolimus, mycophenolate mofetil, an immunosuppressive antiserum, an immunosuppressive antibody, or a combination thereof of the agents. In particular aspects, the therapeutic agent(s) is the immunosuppressive antisera ATG, Atgam, Thymoglobulin, the immunosuppressive antibody OKT3 or a combination thereof. In other embodiments, the therapeutic agent(s) comprise IL-4.
The invention provides at least one therapeutic cocktail comprising a combined effective amount of IL-4 and at least one mutant IL-10 polypeptide that comprises a substantially mammalian IL-10 amino acid sequence wherein isoleucine at position 87 of the mature polypeptide(s) is replaced by alanine or glycine.
The invention provides a method for inducing immunosuppression, comprising contacting a population of immune effector cells with a biologically effective amount of at least a first mutant IL-10 polypeptide that comprises a substantially mammalian IL-10 amino acid sequence wherein isoleucine at position 87 of the mature polypeptide(s) is replaced by alanine or glycine. In certain embodiments, the activity of T cell(s) or NK cell(s) is down-regulated. In certain aspects, the T cell(s) or NK cell(s) cytokine production is inhibited. In other aspects, T cell anergy is induced in one or more T-cell(s). In certain embodiments, the activity of B cell(s) or antigen presenting cell(s) is down-regulated. In further aspects, one or more monocyte, macrophage or dendritic cell(s)"" cytokine production is inhibited.
In certain embodiments, the proliferation of T cell(s), B cell(s) or antigen presenting cell(s) is inhibited. In other embodiments, the migration of immune effector cell(s) is inhibited. In further embodiments, the population of immune effector cell(s) is comprised within an animal and the mutant IL-10 polypeptide(s) is provided to the animal(s). In additional embodiments, the mutant IL-10 polypeptide(s) is provided by administering the polypeptide(s) to the animal(s).
In other embodiments, the mutant IL-10 polypeptide(s) is provided by administering to the animal(s) at least one gene that expresses the polypeptide(s). In certain aspects, the mutant IL-10 polypeptide(s) is provided by administering to the animal(s) at least one recombinant virus comprising an exogenous gene that expresses the polypeptide(s). In other aspects, the mutant IL-10 polypeptide(s) is provided by administering to the animal(s) at least one recombinant cell comprising an exogenous gene that expresses the polypeptide(s). In further aspects, the recombinant cell(s) is prepared by providing the exogenous gene(s) that expresses the polypeptide(s) to at least one cell obtained from the animal(s); and re-administering the recombinant cell(s) to the same animal(s). The cell(s) may be administered to the same animal from which they were obtained.
In certain aspects, the animal(s) has or is at risk of developing at least one inflammatory disease. In certain instances, the animal(s) has or is at risk of developing at least one chronic inflammatory disease. In other instances, the animal(s) has or is at risk of developing at least one autoimmune inflammatory disease. In additional instances, the animal(s) has or is at risk of developing at least one vascular inflammatory disease. In some instances, the animal(s) has or is at risk of developing inflammatory bowel disease. In particular instances, the animal(s) has or is at risk of developing contact hypersensitivity or delayed type hypersensitivity.
In certain aspects, the animal(s) has or is at risk of developing endotoxin-induced toxicity. In other aspects, the animal(s) has or is at risk of developing psoriasis. In some aspects, the animal(s) has or is at risk of developing ischemia-reperfussion injury. In particular aspects, the animal(s) has or is at risk of developing at least one neurological disease. In further aspects, the animal(s) has at least one tumor and is in need of inhibiting tumor immunity. In additional aspects, the animal(s) is undergoing or awaiting a skin graft. In specific aspects, the animal(s) is undergoing or awaiting at least one organ transplant. In particular instances, the animal(s) is undergoing or awaiting at least one kidney, liver or heart transplant, or combination thereof.
In certain aspects, the animal(s) is further provided with at least one biologically effective amount of at least a second therapeutic agent. In certain instances, the animal(s) is further provided with at least one biologically effective amount of at least a second immunosuppressive agent. In additional aspects, the animal(s) is further provided at least a third, at least a fourth, at least a fifth, comprise at least a sixth, at least a seventh or more agents. In some facets, the animal(s) is further provided with at least one biologically effective amount of one or more of the following agent(s): a corticosteroid, sulfasalazine, cyclosporin A, mercaptopurine, azathioprine, tacrolimus, sirolimus, mycophenolate mofetil, an immunosuppressive antiserum, an immunosuppressive antibody, IL-4 or combination thereof. In additional facets, the animal(s) is further provided with at least one biologically effective amount of one or more of the following agents: the immunosuppressive antisera ATG, Atgam, Thymoglobulin, the immunosuppressive antibody OKT3 or a combination thereof.
In certain aspects, the mutant IL-10(s) is provided to the animal(s) parenterally. In certain instances, the mutant IL-10(s) is provided to the animal(s) sublingually, via intravenous administration or via a combination thereof. In certain aspects, the animal(s) is at least one human subject.
The invention provides a method for inducing immunosuppression in the absence of significant immunostimulation, comprising contacting one or more population(s) of immune effector cell(s) with one or more separate amount(s) of at least a first mutant IL-10 polypeptide effective to induce immunosuppression in the absence of significant T cell or B cell stimulation or proliferation, the mutant IL-10 polypeptide(s) comprising a substantially mammalian IL-10 amino acid sequence wherein isoleucine at position 87 of the mature polypeptide is replaced by alanine or glycine.
The invention provides a method for inducing immunosuppression in one or more animal(s) in need of immunosuppression, comprising administering to the animal(s) at least one therapeutically effective amount of at least a first mutant IL-10 polypeptide that comprises a substantially mammalian IL-10 amino acid sequence wherein isoleucine at position 87 of the mature polypeptide is replaced by alanine or glycine. In certain embodiments, the animal(s) hasp or is at risk of developing at least one inflammatory disease. In certain aspects, the animal(s) has or is at risk of developing inflammatory bowel disease. In certain embodiments, the animal(s) has or is at risk of developing ischemia-reperfussion injury. In other embodiments, the animal(s) has at least one tumor and is in need of inhibiting tumor immunity. In some embodiments, the animal(s) is undergoing or awaiting at least one skin graft. In additional embodiments, the animal(s) is undergoing or awaiting at least one organ transplant. In particular embodiments, an at least a second therapeutic agent is further administered to the animal(s). In certain aspects, an at least a second immunosuppressive agent is further administered to the animal(s). In additional aspects, at least a third, at least a fourth, at least a fifth, comprise at least a sixth, at least a seventh or more agents is further administered to the animal(s). In certain instances, at least one therapeutically effective amount of one or more of the following agent(s): corticosteroid, sulfasalazine, cyclosporin A, mercaptopurine, azathioprine, tacrolimus, sirolimus, mycophenolate mofetil, an immunosuppressive antiserum, an immunosuppressive antibody or combination thereof is further administered to the animal(s). In certain facets, at least one therapeutically effective amount of one or more of the following agent(s): the immunosuppressive antisera ATG, Atgam, Thymoglobulin, the immunosuppressive antibody OKT3 or combination thereof is further administered to the animal(s). In certain instances, at least one therapeutically effective amount of IL-4 is further administered to the animal(s). In other instances, an at least a second therapeutic agent is administered to the animal(s) simultaneously with the mutant IL-10 polypeptide(s). In some instances, the at least a second therapeutic agent is administered to the animal(s) sequentially to the mutant IL-10 polypeptide(s). In certain embodiments, the animal(s) is at least one human subject.
The invention provides a method of treating at least one animal having or at risk of developing inflammatory disease or condition, comprising administering to the animal(s) a therapeutically effective amount of at least a first mutant IL-10 polypeptide that comprises a substantially mammalian IL-10 amino acid sequence wherein isoleucine at position 87 of the mature polypeptide is replaced by alanine or glycine.
The invention provides a method of treating at least one animal having or at risk of developing at least one inflammatory bowel disease, comprising administering to the animal(s) at least one therapeutically effective amount of at least a first mutant IL-10 polypeptide that comprises a substantially mammalian IL-10 amino acid sequence wherein isoleucine at position 87 of the mature polypeptide is replaced by alanine or glycine. In certain embodiments, the inflammatory bowel disease is Crohn""s Disease. In other embodiments, the inflammatory bowel disease is ulcerative colitis. In some embodiments, at least one therapeutically effective amount of at least a second therapeutic agent is further administered to the animal(s). In additional aspects, at least a third, at least a fourth, at least a fifth, comprise at least a sixth, at least a seventh or more agents is further administered to the animal(s). In particular embodiments, the animal(s) is at least one human subject.
The invention provides a method of treating at least one animal undergoing or awaiting at least one graft or transplant, comprising administering to the animal(s) at least one therapeutically effective amount of at least a first mutant IL-10 polypeptide that comprises a substantially mammalian IL-10 amino acid sequence wherein isoleucine at position 87 of the mature polypeptide is replaced by alanine or glycine. In certain embodiments, the animal(s) is undergoing or awaiting at least one skin graft. In other embodiments, the animal(s) is undergoing or awaiting at least one organ transplant. In particular embodiments, the animal(s) is at least one human subject.
The invention provides a method of treating at least one animal having or at risk of fibrosis, comprising administering to the animal(s) an immunosuppressive amount of at least a first mutant IL-10 polypeptide that comprises at least one substantially mammalian IL-10 amino acid sequence wherein isoleucine at position 87 of the mature polypeptide is replaced by alanine or glycine.
The invention provides a method of treating at least one animal that has at least one tumor and is in need of inhibiting tumor immunity, comprising administering to the animal(s) at least one immunosuppressive amount of at least a first mutant IL-10 polypeptide that comprises a substantially mammalian IL-10 amino acid sequence wherein isoleucine at position 87 of the mature polypeptide is replaced by alanine or glycine. In certain embodiments, at least one therapeutically effective amount of at least a first anti-cancer agent is further administered to the animal(s).
The invention provides a method of treating cancer, comprising administering to at least one animal with at least one tumor, at least one amount of at least a first mutant IL-10 polypeptide effective to induce one or more destructive immune response(s) against the tumor(s), the mutant IL-10 polypeptide(s) comprising a substantially mammalian IL-10 amino acid sequence wherein isoleucine at position 87 of the mature polypeptide is replaced by alanine or glycine. In certain embodiments, the animal(s) is at least one human subject.
In keeping with the use of longstanding patent terminology, the terms xe2x80x9caxe2x80x9d or xe2x80x9canxe2x80x9d, when used with the term xe2x80x9ccomprisingxe2x80x9d, xe2x80x9ccomprisesxe2x80x9d, xe2x80x9cincludesxe2x80x9d or xe2x80x9cincludingxe2x80x9d, may mean one or more than one herein the specification and claims.
Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.